CN101732704A - Method for preparing inactivated bivalent vaccine against rabbit hemorrhagic disease and pasteurella multocida disease - Google Patents
Method for preparing inactivated bivalent vaccine against rabbit hemorrhagic disease and pasteurella multocida disease Download PDFInfo
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Abstract
The invention provides a method for preparing an inactivated bivalent vaccine against a rabbit hemorrhagic disease and a pasteurella multocida disease, which comprises the following steps: inoculating a rabbit hemorrhagic disease strain CD85-2 strain to a rabbit, aseptically collecting the heart, liver, spleen, lung or kidney of the rabbit dead in 24 to 96h, and removing the connective tissues on the heart, liver, spleen, lung or kidney; preparing the mashed and filtered heart, liver, spleen, lung or kidney into an emulsion after mashing and filtering, and inactivating the emulsion; reactivating a rabbit originated capsula group A pasteurella multocida strain C51-17 strain, performing propagation culture on the reactivated strain, and inactivating the liquid C51-17 strain containing the A type pasteurella multocida strain obtained by culture; and mixing the inactivated liquid strain and an adjuvant, concentrating the mixture to obtain the liquid strain containing the A type pasteurella multocida C51-17, mixing the liquid strain and the inactivated emulsion to obtain the inactivated bivalent vaccine. The inactivated bivalent vaccine prepared by the method has the advantages of high safety, high vaccination efficacy and long shelf life.
Description
Technical field
The present invention relates to a kind of method for preparing rabbit hemorrhagic disease, pasteurella multocida disease bivalent inactivated vaccine.
Background technology
Rabbit hemorrhagic disease be 1984 first in the deadly infectious disease of a kind of rabbit of China southern area of Jiangsu Province outburst.Young and the adult rabbit of main harm, mortality rate be up to more than 90%, and not pathogenic to rabbit childhood below 2 monthly ages.This sick pathological change is hemorrhage with each organa parenchymatosum of whole body to be feature, especially obvious with trachea, lung, liver, kidney.This disease epidemic period uses each antibiotics in the warren, epidemic-stricken area, and sulfa drugs is treated, and all fails to reduce M ﹠ M.At present obtained the good rabbit hemorrhagic disease virus CD of a strain immunogenicity in epidemic-stricken area collection, separation, evaluation, selection-breeding
85-2Strain is used for the production of inactivated vaccine, and using in the area, Sichuan has the excellent prevention effect.
The rabbit Bacillus pasteurii disease is the hueppe's disease of the rabbit that caused by the rabbit pasteurella multocida.The serotype that causes the pasteurella multocida of rabbit hueppe's disease death is the A type, and each manufacturer production of the whole nation prevents this sick vaccine more at present, uses bacterial strain widely different.Rabbit source capsular serotype A group pasteurella multocida C by China Veterinery Drug Inspection Office's evaluation, keeping, supply
51-17Do to produce and use strain, facts have proved that the immunogenicity of this strain is good.Yet,, usually there are problems such as biological safety is poor, side reaction big, unstable product quality with the vaccine that is prepared from after virus stain or the bacterial strain deactivation according to the preparation method of existing vaccine.A lot of in the world at present countries have stopped using inactivated vaccine, also forbid from using the national import livestock products of inactivated vaccine.
Along with the development of rabbit keeping scale, intensification, other several big obstruction rabbit keeping development infectious diseases are still popular, as the sound development of serious threat rabbit keeping such as Bacillus pasteurii disease, clostridieum welchii disease, caused enormous economic loss.In order to satisfy the needs of rabbit keeping diseases prevention and treatment, need develop at Bacillus pasteurii disease and rabbit hemorrhagic disease and be effective vaccine.
Summary of the invention
A purpose of the present invention has provided a kind of method for preparing the good rabbit hemorrhagic disease of safety, pasteurella multocida disease bivalent inactivated vaccine.
The invention provides a kind of method for preparing rabbit hemorrhagic disease, pasteurella multocida disease bivalent inactivated vaccine, this method may further comprise the steps:
A. at first with rabbit poison hemorrhage strain CD
85-2Be inoculated in rabbit, gather inoculation back 24-96 hour heart with interior dead rabbit, liver, spleen, lung or kidney, and reject the connective tissue on the heart, liver, spleen, lung or the kidney;
B. be made into Emulsion after the heart, liver, spleen, lung or the kidney that will reject the dead rabbit of connective tissue smashed to pieces, filtered, and this Emulsion is carried out deactivation;
C. with rabbit source capsular serotype A group pasteurella multocida C
51-17Bacterial strain brings back to life, and the bacterial strain after will bringing back to life again carries out enrichment culture, contains rabbit source capsular serotype A group pasteurella multocida C with what cultivation obtained
51-17Bacterium liquid carries out deactivation;
D. the bacterium liquid after the deactivation that step c is obtained mixes with adjuvant, and is static, obtains containing rabbit source capsular serotype A group pasteurella multocida C
51-17Bacterium liquid;
E. deactivation Emulsion that step b is obtained and steps d obtain contains rabbit source capsular serotype A group pasteurella multocida C
51-17Bacterium liquid mix, obtain rabbit hemorrhagic disease, pasteurella multocida disease bivalent inactivated vaccine.
Preferably, further comprising the steps of between described steps d and step e: with the described rabbit source capsular serotype A group pasteurella multocida C that contains
51-17Bacterium liquid concentrate, make rabbit source capsular serotype A group pasteurella multocida C in this bacterium liquid
51-17Concentration be 1.5 times before concentrating.
Bacterium liquid after the deactivation concentrates, and makes A type pasteurella multocida C
51-17Concentration be 1.5 times before concentrating.
Rabbit hemorrhagic disease virus CD among the present invention
85-2Be the inventor from Sichuan Yingjing, the tenth of the twelve Earthly Branches sun, four epidemic-stricken areas in Yaan and suburb, Chengdu gather back several natural infection poison, identify the bacterial strain that the virulence that filters out is strong, immunogenicity good, the blood clotting valency is high from blood clotting valency, specificity, virulence, immunogenicity, pure property aspect.Described rabbit hemorrhagic disease virus (latin name: CD Rabbit hemorrhagicdisease virus)
85-2Its deposit number is CGMCC No.2705, (CGMCC, Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101) carried out the biomaterial preservation to this virus stain at China Committee for Culture Collection of Microorganisms common micro-organisms center on October 14th, 2008.
The serotype that causes the pasteurella multocida of rabbit hueppe's disease death is the A type, and each manufacturer production of the whole nation prevents this sick vaccine more at present, uses bacterial strain widely different.Described rabbit source capsular serotype A group pasteurella multocida (latin name: C Rabbit Pasteurella.multocida capsularserotypes A)
51-17Bacterial strain, its deposit number is CGMCC No.2704, (CGMCC, Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101) carried out the biomaterial preservation to this bacterial strain at China Committee for Culture Collection of Microorganisms common micro-organisms center on October 14th, 2008.Described rabbit source capsular serotype A group pasteurella multocida abbreviate the rabbit pasteurella multocida as.
Preferably, further comprising the steps of after described steps d or step e: as to contain rabbit source capsular serotype A group pasteurella multocida C to what steps d obtained
51-17Bacterium liquid or the mixed bacteria liquid that obtains of step e in add antiseptic.
This antiseptic is preferably thimerosal.
Preferably, the heart of smashing rabbit to pieces, liver, spleen, lung or the kidney and the 50-10 that contain 0.05-0.07g/ head part in rabbit hemorrhagic disease, the pasteurella multocida disease bivalent inactivated vaccine
3The rabbit source capsular serotype A group pasteurella multocida C of hundred million bacterium/head parts
51-17In this inactivated vaccine, the amount of every part is 1ml.Because the amount to the heart, liver, spleen, lung or the kidney of smashing rabbit to pieces in this vaccine has requirement, in this vaccine, in 0.05-0.07g/ head part scope, just can meet the demands as long as smash the amount of the heart, liver, spleen, lung or the kidney of rabbit to pieces.Same for the rabbit source capsular serotype A group pasteurella multocida C in this vaccine
51-17, as long as the amount of this strain is at 50-10
3Hundred million bacterium/head parts just can meet the demands.
Preferably, described step a is: with rabbit hemorrhagic disease strain CD
85-2Do 10 times of dilutions with normal saline, the rabbit of inoculation body weight 1.5-2.0kg, every subcutaneous injection 1ml, aseptic collection inoculation back 24-96 hour the heart, liver, spleen, lung or kidney with histopathology variation with rabbit in 1 hour of interior death time.
Preferably, aseptic collection inoculation back 24-96 hour liver or spleen with histopathology variation with rabbit in 1 hour of interior death time.
Preferably, rabbit is selected from the negative rabbit of rabbit poison hemorrhage antibody of above 1.5-2.0kg body weight of 2 monthly ages.The age of the experimental rabbit among the present invention if more than 2 monthly ages all can, even old rabbit is unaffected.
Rabbit hemorrhagic disease virus does not infect other animals only to above rabbit easy infection of 2 monthly ages, is difficult to simultaneously make a breakthrough aspect cell culture, therefore adopts rabbit hemorrhagic disease strain CD
85-2Virus of proliferation is produced virus with the susceptible rabbit, its standard be from Pest-or disease-free area without immunity, body weight is the healthy negative antibody rabbit of 1.5kg-2.0kg.By in the large-scale production of breadboard test and pilot scale, adopt the rabbit of this requirement to produce virus always, it is stable to produce virus technology, and the virus multiplication titre can remain on 2 all the time
10More than individual bacterium/ml.
Preferably, step b is that the heart, liver, spleen, lung or the kidney that will reject the dead rabbit of connective tissue are smashed to pieces with normal saline, is made into Emulsion after the filtration, and the heart of smashing to pieces in the Emulsion, liver, spleen, lung or kidney and normal saline weight ratio are 1: 9.
Preferably, step b added the formaldehyde by Emulsion weight 0.4%, at 30-40 ℃ of deactivation 36-48 hour.
More preferably, 37 ℃ of deactivations 36 hours, when deactivation, stirred once in every interval 4-6 hour.
The inventor is respectively 0.2%, 0.3%, 0.4% and 0.5% formalin with concentration, and 37 ℃ of deactivations 36,48 hours, deactivation finish the back and do deactivation with two susceptible rabbit and check.When concentration of formaldehyde was 0.2-0.3%, 37 ℃ of deactivations 36 or 48 hours can not be with viral complete inactivations through the deactivation testing identity; When concentration of formaldehyde was 0.4%, the inoculation rabbit did not have any untoward reaction, proves certain deactivation; When concentration of formaldehyde was 0.5%, the part rabbit had the of short duration phenomenon of stopping eating, and showed that formaldehyde is excessive.Therefore adopting Emulsion weight is that 0.4% formaldehyde was 37 ℃ of deactivations 36 hours.
Preferably, step c is with rabbit source capsular serotype A group pasteurella multocida C
51-17Inoculation is in the martin's bouillon that contains 0.1% cracking blood cell whole blood, cultivate for 37 ℃ and brought back to life in 24 hours, again with the inoculation of bringing back to life in the improvement Martin Agar Plating enrichment culture that contains 0.1% cracking blood cell whole blood and 4% healthy animal serum, under 36-37 ℃, enrichment culture 18-24 hour.
Preferably, in steps d, contain rabbit source capsular serotype A group pasteurella multocida C to what propagation obtained
51-17Bacterium liquid in add the formaldehyde of its gross weight 0.15%, at 30-40 ℃ of deactivation 10-15 hour.
More preferably 37 ℃ of deactivations 12 hours.
A type pasteurella multocida liquid through being up to the standards confirms to add formalin by 0.15% of total amount through great number tested data, in 37 ℃ of deactivations 12 hours, stirs for several times therebetween.Deactivation finishes the back and does the deactivation check with Martin's agar, and asepsis growth confirms the deactivation of this bacterium.
Preferably, the adjuvant among the step e is an aluminum salt, and the bacterium liquid after the deactivation and the weight ratio of aluminum salt are 5: 1.
Preferably, the rabbit hemorrhagic disease virus CD among the step a
85-2Rabbit source capsular serotype A group pasteurella multocida C among strain and the step c
51-17Bacterial strain is 3 generations of subculture with interior bacterial strain.
The inventor is by the research and identify of going down to posterity to basic bacterial strain, and to producing the evaluation with bacterial strain, in 7 generations of A type pasteurella multocida basis seed passage number, produces strain and adopts the follow-up generation in 7 generations of going down to posterity to be no more than the strain in 3 generations; Rabbit hemorrhagic disease CD
85-2Basis seed culture of viruses passage number was 6 generations, produced seed culture of viruses and adopted the follow-up generation in 6 generations of going down to posterity to be no more than the strain in 3 generations, can guarantee the immune effect of vaccine.
Unlike the prior art be: the present invention does not directly adopt rabbit hemorrhagic disease strain CD
8 5-2The preparation vaccine, but at first it is inoculated in rabbit, gather inoculation back 24-96 hour heart with interior dead rabbit, liver, spleen, lung or kidney, and the connective tissue on the rejecting heart, liver, spleen, lung or the kidney, be made into Emulsion after the heart, liver, spleen, lung or the kidney of rejecting the dead rabbit of connective tissue smashed to pieces, filter, and this Emulsion of deactivation, with the Emulsion of deactivation and cultivate obtain contain rabbit source capsular serotype A group pasteurella multocida C
51-17Inactivated bacterial liquid mix and to obtain vaccine of the present invention.With respect to directly with rabbit hemorrhagic disease strain CD
85-2With rabbit source capsular serotype A group pasteurella multocida C
51-17The inactivated vaccine of making after the deactivation, the safety of the vaccine that according to said method obtains is better, also can guarantee the immune effect of vaccine simultaneously.Carry out rabbit hemorrhagic disease that the present invention adopts the inventive method to obtain, A type pasteurella multocida disease bivalent inactivated vaccine through safety examination: subcutaneous or intramuscular inoculation is in the easy infection rabbit at 1 monthly age, rabbit contrast with the inoculation normal saline, the rabbit mental status of inoculation is no abnormal, appetite normal, the body temperature no change, and the injection site has no adverse reaction.Be inoculated in conceived healthy female rabbit simultaneously and carry out the safety detection, the result shows the pregnant female rabbit after the immunity, through observing, the mental status of the female rabbit of gestation is no abnormal, appetite normal, the body temperature no change, the injection site has no adverse reaction, farrowing is normal, and therefore the vaccine that is prepared from by the inventive method is safe.
The immune efficacy check of the vaccine that the inventive method obtains: 6 batches of laboratory products that are prepared from by the inventive method immunity respectively carry out immunity through the antibody test all negative healthy weaned newborn rabbit and the rabbit that grows up, respectively the weaned newborn rabbit and the rabbit that grows up are carried out counteracting toxic substances after the immunity, its result shows: weaned newborn rabbit behind the vaccine, the rabbit hemorrhagic disease protective rate is 100%, the rabbit pasteurella multocida is had 75% protective rate; With the adult rabbit of this vaccine immunity, the rabbit hemorrhagic disease protective rate is 100%; The rabbit pasteurella multocida there is protective rate more than 75%.
About the duration of immunity of vaccine, carry out immune period test in the warren with the prepared in laboratory vaccine, the result shows the duration of immunity of this vaccine: rabbit hemorrhagic disease is 12 months, the sick duration of immunity of rabbit pasteurella multocida 5 months.This can satisfy clinically the immune requirement to rabbit.
About the storage life of vaccine, leave the vaccine that adopts the inventive method to be prepared from 2-8 ℃ of shady place reach 12 months and 20-25 ℃ of condition under deposited 10 days.With 1 using dosage the healthy susceptible rabbit of 1.5-2.0kg is carried out immunity, carry out the counteracting toxic substances protection test, all be protected, this confirms that fully vaccine quality does not change in above-mentioned preservation condition and storage life.
About the minimum immune dosage of vaccine, the inventor has carried out the mensuration of vaccine to the minimum immune dosage of rabbit in development process, establishes corresponding non-immunize rabbit simultaneously in contrast.The immunity back was carried out the counteracting toxic substances protection test respectively in 14,21 days, and the result confirms that this vaccine minimum immune dosage is 0.5ml.The convenience of consideration clinical injection and production cost, immune effect guarantee, this vaccine using dosage is decided to be 1ml.
Description of drawings
Rabbit hemorrhagic disease virus provided by the invention (latin name: CD Rabbit hemorrhagicdisease virus)
85-2Its deposit number is CGMCC No.2705, (CGMCC, Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101) carried out the biomaterial preservation to this virus stain at China Committee for Culture Collection of Microorganisms common micro-organisms center on October 14th, 2008.
Described rabbit source capsular serotype A group pasteurella multocida (latin name: C Rabbit Pasteurella.multocida capsular serotypes A)
51-17Bacterial strain, its deposit number is CGMCC No.2704, (CGMCC, Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101) carried out the biomaterial preservation to this bacterial strain at China Committee for Culture Collection of Microorganisms common micro-organisms center on October 14th, 2008.
Fig. 1 represents to prepare the flow chart of rabbit hemorrhagic disease of the present invention, pasteurella multocida disease bivalent inactivated vaccine.
The specific embodiment
Embodiment 1
Rabbit poison hemorrhage CD
85-2The cultivation of going down to posterity of strain
With the rabbit hemorrhagic disease strain CD after the preservation
85-2Do 10 times of dilutions with normal saline, be inoculated in for two monthly ages, from Pest-or disease-free area without immunity, body weight is the healthy negative antibody rabbit of 1.5kg.Every subcutaneous injection 1ml, the virus titer in this rabbit can remain on 2
10More than individual bacterium/ml.The rabbit of dead body weight 1.5kg in 24-96 hour after the aseptic collection inoculation, the aseptic collection death time is no more than 1 hour rabbit and has the heart, liver, lung, spleen or the kidney that histopathology changes, and reject connective tissue on it, select liver as seed culture of viruses in this example.
Adopt the strain CD that passed for 3 generations
85-2Inoculation, the heart, liver, lung, spleen or the kidney of the dead rabbit of connective tissue will be rejected, preferred liver adds normal saline, and to smash, filter the volume ratio that is mixed with the internal organs smashed to pieces and normal saline to pieces be 1: 9 the Emulsion of organizing, add concentration more respectively and be 0.2%, 0.3%, 0.4% and 0.5% formalin and carry out deactivation, the deactivation condition is: 37 ℃ of deactivations 36 hours, stirred once in 4-6 hour at interval, deactivation finishes the back and does the deactivation check with two susceptible rabbit.When concentration of formaldehyde was 0.2-0.3%, 37 ℃ of deactivations 36,48 hours can not be with viral complete inactivation through the deactivation testing identity; When concentration of formaldehyde was 0.4%, the inoculation rabbit did not have any untoward reaction, proves certain deactivation; When concentration of formaldehyde was 0.5%, the part rabbit had the of short duration phenomenon of stopping eating, and showed that formaldehyde is excessive.So the deactivation condition of selecting is: with concentration is that 0.4% formaldehyde carries out deactivation, and the deactivation temperature is 37 ℃, the inactivation time that continues 36 hours, and when deactivation every stirring in 4 hours once, obtain the Emulsion of deactivation.
Rabbit source capsular serotype A group pasteurella multocida C with preservation
51-17Inoculation is in the martin's bouillon that contains 0.1% cracking blood cell whole blood, cultivate for 37 ℃ and brought back to life in 24 hours, again with the inoculation of bringing back to life in the improvement Martin Agar Plating enrichment culture that contains 0.1% cracking blood cell whole blood and 4% healthy animal serum, under 36 ℃, enrichment culture 18 hours.The martin's bouillon that contains 0.1% cracking blood cell whole blood in the present embodiment is available from school of life and health sciences Qingdao, Qingdao GaoKeYuan Hai Bo biotech company, commodity improvement by name Martin fluid medium.Contain 0.1% the cracking blood cell whole blood and the improvement Martin Agar Plating of 4% healthy animal serum and prepared voluntarily by our unit, its prescription is as follows: improvement martin's bouillon 1000ml, 5g peptone, 10g agar.
Contain rabbit source capsular serotype A group pasteurella multocida C to what enrichment culture obtained
51-17The formaldehyde that adds its gross weight 0.15% in the bacterium liquid was 37 ℃ of deactivations 12 hours.Deactivation finishes the back and does the deactivation check with Martin's agar, asepsis growth, confirm deactivation, bacterium liquid 5 parts by volume after the deactivation are added sterilization aluminum salt 1 parts by volume, static leaving standstill 2-3 day removed supernatant, and it is concentrated, the concentration of rabbit source capsular serotype A group pasteurella multocida after concentrating in the bacterium liquid is 1.5 times of rabbit source capsular serotype A group pasteurella multocida concentration before concentrating, and is system vaccine bacterium liquid antigen.In this bacterium liquid antigen, add long-pending than the CD that is 1: 1 again with respect to rabbit source capsular serotype A group pasteurella multocida bacteria liquid
85-2Deactivation Emulsion, add thimerosal by 0.01% of bacterium liquid and Emulsion gross weight, stir, make rabbit hemorrhagic disease, rabbit Bacillus pasteurii disease bivalent inactivated vaccine.Through check, contain the internal organs of smashing rabbit to pieces of 0.05g/ head part and the rabbit source capsular serotype A group pasteurella multocida C of 5,000,000,000 bacterium/head parts in this vaccine
51-17
We have produced six batches of laboratory products and the six batches of clinical trials with vaccine totally 150 ten thousand parts by above technology, and qualification rate 100% confirms that this technology is to improve and feasible.
Embodiment 2
With the rabbit hemorrhagic disease strain CD after the preservation
85-2Do 10 times of dilutions with normal saline, be inoculated in for 5 ages, from Pest-or disease-free area without immunity, body weight is the healthy negative antibody rabbit of 2.0kg.Every subcutaneous injection 1ml, the virus titer in this rabbit can remain on 2
10More than.The rabbit of dead body weight 2.0kg in 24-96 hour after the aseptic collection inoculation, the aseptic collection death time is no more than 1 hour rabbit and has the heart, liver, lung, spleen or the kidney that histopathology changes, and reject connective tissue on it, select spleen as seed culture of viruses in this example.
Adopt the strain CD that passed for 3 generations
85-2Inoculation, the heart, liver, lung, spleen or the kidney of the dead rabbit of connective tissue will be rejected, selecting spleen to add normal saline, to smash, filter the volume ratio that is mixed with the internal organs smashed to pieces and normal saline to pieces be 1: 9 the Emulsion of organizing, add concentration more respectively and be 0.2%, 0.3%, 0.4% and 0.5% formalin and carry out deactivation, the deactivation condition is: 37 ℃ of deactivations 36 hours, stirred once in 4-6 hour at interval, deactivation finishes the back and does the deactivation check with two susceptible rabbit.When concentration of formaldehyde was 0.2-0.3%, 37 ℃ of deactivations 36,48 hours can not be with viral complete inactivation through the deactivation testing identity; When concentration of formaldehyde was 0.4%, the inoculation rabbit did not have any untoward reaction, proves certain deactivation; When concentration of formaldehyde was 0.5%, the part rabbit had the of short duration phenomenon of stopping eating, and showed that formaldehyde is excessive.So the deactivation condition of selecting is: with concentration is that 0.4% formaldehyde carries out deactivation, and the deactivation temperature is 37 ℃, the inactivation time that continues 36 hours, and when deactivation every stirring in 6 hours once, obtain the Emulsion of deactivation.
Rabbit source capsular serotype A group pasteurella multocida C with preservation
51-17Inoculation is in the martin's bouillon that contains 0.1% cracking blood cell whole blood, cultivate for 37 ℃ and brought back to life in 24 hours, again with the inoculation of bringing back to life in the improvement Martin Agar Plating enrichment culture that contains 0.1% cracking blood cell whole blood and 4% healthy animal serum, under 36 ℃, enrichment culture 18 hours.The martin's bouillon that contains 0.1% cracking blood cell whole blood in the present embodiment is available from the commodity improvement by name Martin fluid medium of school of life and health sciences Qingdao, Qingdao GaoKeYuan Hai Bo biotech company.Contain 0.1% the cracking blood cell whole blood and the improvement Martin Agar Plating of 4% healthy animal serum and prepare voluntarily, its prescription is as follows: improvement martin's bouillon 1000ml, 5g peptone, 10g agar.
To the rabbit source capsular serotype A group pasteurella multocida C that enrichment culture obtains
51-17The formaldehyde that adds its gross weight 0.15% in the bacterium liquid was 37 ℃ of deactivations 12 hours.Deactivation finishes the back and does the deactivation check with Martin's agar, asepsis growth, confirm deactivation, bacterium liquid 5 parts by volume after the deactivation are added sterilization aluminum salt 1 parts by volume, static leaving standstill 2-3 day removed supernatant, and it is concentrated, the concentration of rabbit source capsular serotype A group pasteurella multocida after concentrating in the bacterium liquid is 1.5 times before concentrating, and is system vaccine bacterium liquid antigen.0.02% of its weight of adding thimerosal in this bacterium liquid antigen adds long-pending than the CD that is 1: 1 with respect to A type pasteurella multocida bacteria liquid again in this bacterium liquid antigen again
85-2Deactivation Emulsion, stir, make rabbit hemorrhagic disease, rabbit Bacillus pasteurii disease bivalent inactivated vaccine.Through check, contain the heart of smashing rabbit to pieces, liver, spleen, lung or kidney of 0.05g/ head part and the rabbit source capsular serotype A group pasteurella multocida C of 5,000,000,000 bacterium/head parts in this vaccine
51-17
We have produced six batches of laboratory products and the six batches of clinical trials with vaccine totally 150 ten thousand parts by above technology, and qualification rate 100% confirms that this technology is to improve and feasible.
Embodiment 3
The safety examination of vaccine
Will according to embodiment 1 method or the vaccine that obtains be subcutaneous or muscle single dose 1ml inoculates 1 monthly age healthy susceptible rabbit and carries out safety testing: with the vaccine of trial-production subcutaneous or 1 monthly age of intramuscular inoculation each 4 of healthy susceptible rabbit, 1ml/ only, inject the non-immunity contrast of 1ml/ sterile saline only in addition simultaneously to 4 rabbit, measure body temperature, observed 10.After the inoculation, the mental status is no abnormal as a result, and appetite is normal, and body temperature does not have significant change, untoward reaction such as injection site NIP.
Subcutaneous or muscle single dose 1ml 1 monthly age of repeated inoculation healthy susceptible rabbit safety testing: with the vaccine of trial-production subcutaneous or 1 monthly age of intramuscular inoculation each 4 of healthy susceptible rabbit, 1ml/ only, repeated inoculation once after 7 days, other establishes, and 4 rabbit are injected simultaneously and the non-immunity contrast of a duplicate injection 1ml/ sterile saline, measure body temperature, observed 10.After the inoculation, the mental status is no abnormal as a result, and appetite is normal, and body temperature does not have significant change, untoward reaction such as injection site NIP.
Subcutaneous or muscle overdose 4ml inoculates the safety testing of healthy susceptible rabbit of 1 monthly age: with the vaccine of trial-production subcutaneous or 1 monthly age of intramuscular inoculation each 4 of healthy susceptible rabbit, 4ml/ only, other establishes the non-immunity contrast that 4 rabbit are injected 4ml/ sterile saline simultaneously, measures body temperature, observes 10.After the inoculation, the mental status is no abnormal as a result, and appetite is normal, and body temperature does not have significant change, untoward reaction such as injection site NIP.
Subcutaneous or muscle two multiple dose 2ml inoculate the safety testing of 10-15 days female rabbits of conceived healthy susceptible gestation: the subcutaneous or pregnant 10-15 of intramuscular inoculation days healthy susceptible rabbit are each 4 with the vaccine of trial-production, 2ml/ only, other establishes the non-immunity contrast of 4 whiles and a duplicate injection 1ml/ sterile saline, measure body temperature, observe farrowing situation etc.After the inoculation, the mental status is no abnormal as a result, and appetite is normal, and body temperature does not have significant change, untoward reaction such as injection site NIP, and the farrowing situation is normal.
Above result of the test shows that this vaccine is safe.
Embodiment 4
The check of vaccine potency
6 batches of laboratory products (0201001,0202002,0202003,0202004,0202005,0202006) immunity respectively reach each the 4/group of rabbit of growing up through all negative healthy weaned newborn rabbit of antibody test, 1ml/ only, immunity back 14,21 days, contrast rabbit that condition is identical and weaned newborn rabbit and the rabbit that grows up carry out counteracting toxic substances together respectively, its result: 0201001-0202006 criticizes vaccine immune weaned newborn rabbit respectively, the rabbit hemorrhagic disease protective rate is 100%, and the contrast mortality rate is 15/16; Rabbit pasteurellosis bacillus protective rate is respectively 3/4,3/4,3/4,3/4 promptly 75% protective rate approximately, and the contrast protective rate is 0%.0201001-0202006 criticizes the vaccine adult rabbit of immunity respectively, and the rabbit hemorrhagic disease protective rate is 100%, contrasts dead 14/16; Rabbit pasteurellosis bacillus protective rate is respectively 4/4,3/4,3/4,3/4 protective rate that promptly has more than 75%, and the contrast protective rate is 0%.
We determine in this vaccine product inspection rabbit hemorrhagic disease counteracting toxic substances immunoprotection 4/4 thus; to rabbit pasteurella multocida disease counteracting toxic substances immunoprotection 3/4, so the vaccine that the inventive method is prepared from all has protection to render a service to rabbit hemorrhagic disease and rabbit pasteurella multocida disease.
Embodiment 5
The vaccine immunity phase
6 batches of vaccines (0201001,0202002,0202003,0202004,0202005,0202006) with Laboratory Production carry out immune period test in fixed point from numerous warren, every batch of vaccine divides 5 groups, inoculate 10 1.5-2.0kg susceptible rabbit and 1 monthly age weaned newborn rabbit for every group, 1 using dosage of every subcutaneous injection, each component two parts sets up 5 non-immunize rabbits as blank simultaneously.Through the immunity 120,180,240,300,360 after, do the counteracting toxic substances protection test, its as a result the rabbit hemorrhagic disease immunoprotection be 100% always, 150 days immunoprotections of rabbit Bacillus pasteurii disease still have 75% protective rate.
Therefore stipulate the duration of immunity of this vaccine: rabbit hemorrhagic disease is 12 months, rabbit Bacillus pasteurii disease duration of immunity 5 months.This can satisfy clinically the immune requirement to rabbit.
Embodiment 6
The test of vaccine storage life
Left four batches of laboratory products in 2-8 ℃ of shady place 9 months, 12 months, 15 months, 18 months and 20-25 ℃ of condition under deposited 10 days.With 1 using dosage the healthy susceptible rabbit of 1.5-2.0kg is carried out immunity, after immunity, carried out the counteracting toxic substances protection test respectively in 14,21 days respectively, all be protected, confirm that fully vaccine quality does not change in above-mentioned preservation condition and storage life.Therefore this vaccine is under 2-8 ℃ preservation condition, and storage life is for can reach 12 months.
Embodiment 7
About determining of vaccine minimum immune dosage
We have carried out the mensuration of vaccine to the minimum immune dosage of rabbit in development process, the result confirms: adopt with vaccine with 0.25ml, 0.5ml, four kinds of various dose groups immunity of 0.75ml, 1ml 1.5kg about healthy susceptible rabbit, establish corresponding non-immunize rabbit simultaneously in contrast.The immunity back was carried out the counteracting toxic substances protection test in 14,21 days respectively, its result: 0.25ml group rabbit hemorrhagic disease immunoprotection 3/4, and rabbit pasteurellosis bacillus immunoprotection is 2/4; 0.5ml, 0.75ml, 1ml group rabbit hemorrhagic disease immunoprotection is 4/4, rabbit Bacillus pasteurii disease immunoprotection is 3/4,3/4,3/4.Confirm that by test this vaccine minimum immune dosage is decided to be 0.5ml.
Claims (10)
1. method for preparing rabbit hemorrhagic disease, pasteurella multocida disease bivalent inactivated vaccine, this method may further comprise the steps:
A. at first with rabbit hemorrhagic disease strain CD
85-2Be inoculated in rabbit, gather inoculation back 24-96 hour heart with interior dead rabbit, liver, spleen, lung or kidney, and reject the connective tissue on the heart, liver, spleen, lung or the kidney;
B. be made into Emulsion after the heart, liver, spleen, lung or the kidney that will reject the dead rabbit of connective tissue smashed to pieces, filtered, and this Emulsion is carried out deactivation;
C. with rabbit source capsular serotype A group pasteurella multocida C
51-17Bacterial strain brings back to life, and the bacterial strain after will bringing back to life again carries out enrichment culture, contains rabbit source capsular serotype A group pasteurella multocida C with what cultivation obtained
51-17Bacterium liquid carries out deactivation;
D. the bacterium liquid after the deactivation that step c is obtained mixes with adjuvant, and is static, obtains containing rabbit source capsular serotype A group pasteurella multocida C
51-17Bacterium liquid;
E. deactivation Emulsion that step b is obtained and steps d obtain contains rabbit source capsular serotype A group pasteurella multocida C
51-17Bacterium liquid mix, obtain rabbit hemorrhagic disease, pasteurella multocida disease bivalent inactivated vaccine, preferably, further comprising the steps of between described steps d and step e: with the described rabbit source capsular serotype A group pasteurella multocida C that contains
51-17Bacterium liquid concentrate, make rabbit source capsular serotype A group pasteurella multocida C in this bacterium liquid
51-17Concentration be 1.5 times before concentrating.
2. method according to claim 1, wherein further comprising the steps of after described steps d or step e: as to contain rabbit source capsular serotype A group pasteurella multocida C to what steps d obtained
51-17Bacterium liquid or the mixed bacteria liquid that obtains of step e in add antiseptic, this antiseptic is preferably thimerosal.
3. method according to claim 1 and 2 wherein contains the heart of smashing rabbit to pieces, liver, spleen, lung or kidney and the 50-10 of 0.05-0.07g/ head part in the rabbit hemorrhagic disease that obtains of step e, the pasteurella multocida disease bivalent inactivated vaccine
3The rabbit source capsular serotype A group pasteurella multocida C of hundred million bacterium/head parts
51-17
4. according to each described method among the claim 1-3, wherein said step a is: with rabbit hemorrhagic disease strain CD
85-2Do 10 times of dilutions with normal saline, the rabbit of inoculation body weight 1.5-2.0kg, every subcutaneous injection 1ml, aseptic collection inoculation back 24-96 hour the heart, liver, spleen, lung or kidney with histopathology variation with rabbit in 1 hour of interior death time, preferably, aseptic collection inoculation back 24-96 hour liver or spleen with histopathology variation with the rabbit of interior death time in 1 hour.
5. according to each described method among the claim 1-4, rabbit wherein is selected from the negative rabbit of rabbit hemorrhagic disease antibody of above 1.5-2.0kg body weight of 2 monthly ages.
6. according to each described method among the claim 1-5, step b wherein is that the heart, liver, spleen, lung or the kidney that will reject the dead rabbit of connective tissue are smashed to pieces with normal saline, be made into Emulsion after the filtration, the heart of smashing to pieces in the Emulsion, liver, spleen, lung or kidney and normal saline weight ratio are 1: 9.
7. according to each described method among the claim 1-6, wherein step b adds the formaldehyde by Emulsion weight 0.4%, at 30-40 ℃ of deactivation 36-48 hour, stirs once in every interval 4-6 hour during preferably 37 ℃ of deactivations 36 hours and in deactivation.
8. according to each described method among the claim 1-7, wherein step c is with rabbit source capsular serotype A group pasteurella multocida C
51-17Inoculation is in the martin's bouillon that contains 0.1% cracking blood cell whole blood, cultivate for 37 ℃ and brought back to life in 24 hours, again with the inoculation of bringing back to life in the improvement Martin Agar Plating enrichment culture that contains 0.1% cracking blood cell whole blood and 4% healthy animal serum, under 36-37 ℃, enrichment culture 18-24 hour.
9. according to each described method among the claim 1-8, wherein in steps d, contain rabbit source capsular serotype A group pasteurella multocida C to what enrichment culture obtained
51-17Bacterium liquid in add the formaldehyde of its gross weight 0.15%, at 30-40 ℃ of deactivation 10-15 hour, preferably 37 ℃ of deactivations 12 hours.
10. according to each described method among the claim 1-9, wherein the adjuvant among the step e is an aluminum salt, and the volume ratio ratio of bacterium liquid after the deactivation and aluminum salt is 5: 1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102755644A (en) * | 2012-07-31 | 2012-10-31 | 郑州后羿制药有限公司 | Rabbit haemorrhagic disease tissue inactivated vaccine and preparation method thereof |
| CN108904796A (en) * | 2018-07-12 | 2018-11-30 | 江苏省农业科学院 | Rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine and preparation method thereof |
| CN111534461A (en) * | 2020-04-30 | 2020-08-14 | 四川百诺吉科技有限公司 | Rabbit pasteurella multocida enrichment medium and use method thereof |
| CN113046271A (en) * | 2021-04-10 | 2021-06-29 | 福建省农业科学院畜牧兽医研究所 | Rabbit F-type pasteurella multocida and application thereof in preparation of inactivated vaccine |
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2008
- 2008-11-21 CN CN200810227103A patent/CN101732704B/en active Active
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102755644A (en) * | 2012-07-31 | 2012-10-31 | 郑州后羿制药有限公司 | Rabbit haemorrhagic disease tissue inactivated vaccine and preparation method thereof |
| CN102755644B (en) * | 2012-07-31 | 2014-03-26 | 郑州后羿制药有限公司 | Rabbit haemorrhagic disease tissue inactivated vaccine and preparation method thereof |
| CN108904796A (en) * | 2018-07-12 | 2018-11-30 | 江苏省农业科学院 | Rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine and preparation method thereof |
| CN108904796B (en) * | 2018-07-12 | 2022-06-03 | 江苏省农业科学院 | Rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine and preparation method thereof |
| CN111534461A (en) * | 2020-04-30 | 2020-08-14 | 四川百诺吉科技有限公司 | Rabbit pasteurella multocida enrichment medium and use method thereof |
| CN111534461B (en) * | 2020-04-30 | 2023-10-31 | 四川百诺吉科技有限公司 | Rabbit pasteurella multocida enrichment medium and application method thereof |
| CN113046271A (en) * | 2021-04-10 | 2021-06-29 | 福建省农业科学院畜牧兽医研究所 | Rabbit F-type pasteurella multocida and application thereof in preparation of inactivated vaccine |
| CN113046271B (en) * | 2021-04-10 | 2022-10-14 | 福建省农业科学院畜牧兽医研究所 | Rabbit F-type pasteurella multocida and application thereof in preparation of inactivated vaccine |
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|---|---|
| CN101732704B (en) | 2012-08-29 |
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